The p75 neurotrophin receptor (p75NTR), a member of the tumor necrosis factor receptor (TNRF) family, is a 75 kDa cell-surface receptor glycoprotein that binds with similar affinity to the neurotrophin family (brain-derived neurotrophic factor, neurotrophin-3 and neurotrophin-4/5) of growth factors. It was the first receptor described for nerve growth factor (NGF) and shown to facilitate Tyrosine kinase receptor (Trk) signal transduction by the formation of high affinity neurotrophin receptor complexes. In contrast to p75NTR the Trk receptor family binds the neurotrophins with varying specificity resulting in cell survival and process outgrowth.
Although providing a definitive function for p75NTR signalling remains controversial, there is substantial evidence to support the hypothesis that p75NTR can initiate a caspase-mediated, i.e mitochondria-mediated apoptotic pathway in a variety of neural and non-neural cell types. A possible role as a tumor and a metastasis suppressor in tumor cells was recently shown in the capability of p75NTR to suppress growth and nerve growth-factor mediated metastasis of human prostate cancer cells and in the effect of p75NTR expression on the cell survival, proliferation and growth of the human cancer cell line T24. Evidence to support a role for p75NTR as a neuronal death inducing factor is based on experiments wherein an increased cell death upon treatment of various neural cell types such as for example developing chick retinas or cultured sympathetic neurons and proprioceptive neurons with brain-derived neurotrophic factor and/or NGF, can be prevented by application of p75NTR antibodies.
p75NTR has sequence similarity to other TNRF family members both in the cysteine-rich extodomain and in the cytoplasmic sequence known as the death domain. Despite the presence of a death domain in p75NTR there is accumulating evidence that this region does not mediate the ability of p75NTR to promote cell death. Unlike the TNRF death domain the death domain of p75NTR does not interact with other death-domain containing proteins, does not spontaneously multimerize in solution and does not function in the same manner. In fact, it was recently shown that deletion of the death domain sequence has no effect on the ability of p75NTR to kill. Instead of the death domain, the cytoplasmic juxtamembrane region of the p75NTR has been found to be necessary and sufficient to initiate neural cell death. The region was named “Chopper” and shown to induce apoptotic cell death only when bound to the plasma membrane by a lipid anchor.
Apoptosis or programmed cell death is a physiological mechanism to eliminate cells in different tissues during embryogenesis, morphogenesis and cell renewal. Apoptosis is a genetically controlled mechanism that intervenes at advanced and irreversible stages of cell damage. It is accordingly established that apoptosis plays a key role in neuronal death that occurs in some of the major disorders of the CNS such as stroke, Parkinson's disease, Huntington's disease, Alzheimer's disease, Amyotrophic lateral sclerosis (ALS), epilepsy, Spinal Cord Injury (SCI), Multiple Sclerosis (MS), Motor Neuron Disease (MND) and other neurodegenerative diseases (Park et al. 2000; Oh et al. 2000; Lowry et al. 2001; Sedel et al. 1999; Dowling et al. 1999).
In addition to the above, numerous studies show an increased expression of p75NTR after ishemia in brain and heart regions where an massive apoptosis was enregistered. These results suggest that p75NTR may play an important role in neuronal death by post-ischemic apoptosis (Park et al, J. Neuroscience, 2000, 20, 9096-9103).
The p75NTR receptor is also described as cellular signalling partner for Prion and β-amyloid peptides (Della-Bianca et al., J. Biol. Chem. 2001, 276, 38929-38933) and is accordingly involved in the neurotoxic action of these components. These results support the hypothesis that p75NTR would play an important role in the neuronal death observed in prion diseases, i.e. Transmissible spongiform encephalopathies (TSE) and Alzheimer's disease.
Recent studies provide a role for the p75NTR receptor as coreceptor in the signalling pathway of the myelin inhibitory components, i.e. myelin-associated glycoprotein (MAG), Nogo and oligodendrocyte-myelin glycoprotein (Omgp). All of these proteins are localised in the membrane of oligodendrocytes immediately adjacent to the axone and inhibit neuronal growth by binding a common receptor, the Nogo66 receptor (NgR). NgR is linked to the cell surface by means of a glycosylphosphatidylinositol (GPI) anchor but lacks an intracellular signalling domain and accordingly needs p75NTR as signalling partner. It was found that disruption of the NgR signalling complex prevents the inhibitory action of MAG. Hence, as a coreceptor of NgR, p75NTR now emerges as a key player, not only for regulating neuronal development and apoptosis, but also for regulating the inhibition of axon regeneration induced by myelin-associated factors and as such provides a therapeutic target to promote neuronal regeneration.
Although the major role for p75NTR is CNS related, some recent work shows an increased expression of neutrophines and p75NTR with a concommitant apoptosis at lesions caused by atherosclerosis. In addition, for a short variant of p75NTR, that arises from alternative splicing of exon III in the p75NTR locus, it was shown in transgenic mice that the absence of p75NTR leads to a severe phenotype, including partial perinatal lethality and defects in the vascular system. An involvement of neurotrophins and Trk receptors in vasculogenesis has previously been demonstrated; all neurotrophins are detected in the forming of tunic media of the aorta from E13 onward. TrkB and TrkC are expressed in the developing aorta with expression patterns reciprocal to that of p75NTR, and severe heart malformations have been observed in NT3 and in TrkC mutant mice. It thus seems that neurotrophin receptors, now including p75NTR, are essential in the formation of blood vessels. All of these findings suggest a paramount role for p75NTR in vascular pathologies such as for example, atherosclerosis, congenital and rheumatic heart disease, and vascular inflammation
Notwithstanding the recognition of p75NTR as an important therapeutic target present screening methods rely on the interaction of p75NTR with its ligand NGF either in a competitive binding assay using cell membrane preparations of p75NTR expressing cells and radiolabeled NGF as described by Weskamp (Neuron, 1991, 6, 649-663) or by measuring the effect of the compounds to be tested on NGF induced apoptosis in p75NTR expressing cells as described by Tabassum (Int. J. Cancer, 2003, 105, 47-52). Neither method provides the possibility to study the effects of test compounds on the p75NTR signal transduction irrespective of the ligand used.
Present in vitro screens to identify compounds that modulate the p75NTR signalling activity are based on the transient transfection of adherent cell cultures of sensory neurons, rat PC12 cells, 293T cells and wild-type Schwann cells, using DNA constructs that encode for p75NTR or truncated forms of p75NTR that retain the capability to induce apoptotic cell death upon induction with NGF or leukemia inhibitory factor (LIF) (see for example Coulson et al., J. Biol. Chem. 2000, 275, 30537-30545).
Current methods of drug discovery generally involve assessing the biological activity of tens or hundreds of thousands of compounds in order to identify a small number of those compounds having a desired activity against a particular target, i.e. High Throughput Screening (HTS). In a typical HTS related screen format, assays are performed in multi-well microplates, such as 96, 384 or 1536 well plates, putting certain constrains to the setup of the assay to be performed including the availability of the source materials. HTS related screens are preferably performed at room temperature with a single measurement for each of the compounds tested in the assay, requiring short cycle times, with a reproducible and reliable output.
The present invention describes the development of a p75NTR signalling assay that can be performed in an HTS screen format and which is based on a particular transfection method applicable to eukaryotic cells such as for example Hek293T cells.